During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1) and its homologues. NKCC1 belongs to a small gene family with nine homologues in vertebrates. Of these, seven are known to be electroneutral cation-chloride cotransporters while the function of the remaining two vertebrate homologues remains uncertain. NKCC1 is relatively widely expressed in both epithelial and non-epithelial tissues and is known to play important roles in a variety of physiological processes including transepithelial salt and water transport, hearing, olfaction, pain perception, spermatogenesis, and maintenance of blood pressure and vascular tone. [unreadable] [unreadable] NKCC1 is the major Cl entry pathway into salivary acinar cells and thus is primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of the structure/function relationships of this protein and its behavior in acinar cells will greatly improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. NKCC1 is also an important candidate gene for the treatment of salivary hypofunction via gene transfer in our Branch and we are collaborating with Dr. Bruce Baum on some initial experiments related to this. [unreadable] [unreadable] In past studies we have established that the functional unit of NKCC1 is a homodimer and that the intracellular 450 amino acid C-terminus of the protein is largely responsible for its dimerization. We have been employing chemical crosslinking studies and a novel co-immunoprecipitation assay to identify and characterize the amino acids involved in the dimerization interaction. Using these methods we have been able to localize an essential dimerization motif consisting of 5 amino acids; 2 of these residues are NKCC1-specific and result in a homologue specific interaction while the remainder are conserved amongst NKCC1 homologues and appear to form a structural context for the dimerization motif. We have also been able to demonstrate a modest functional interaction between dimer subunits. While the full functional significance of dimer formation is still not established we have been able to show that previous suggestions that NKCC1 forms heterodimers with its homologues to form novel functional units are probably incorrect. The first paper on this project is now in press and we are nearing completion of an additional paper where we are completing the characterization of the 5 amino acid dimerization motif. The consequences of a mutation in one of these amino acids that is associated with human disease are also being investigated.[unreadable] [unreadable] In order to study the dimerization properties of NKCC1 we have developed a specialized co-immunoprecipitation procedure that allows us to gently and specifically elute only one NKCC1 unit from immunoprecipitated NKCC1 dimer pairs. This eluate necessarily includes any other proteins associated with the eluted NKCC1 molecules. We are now using this procedure to screen for proteins that interact with NKCC1, beginning with several candidate proteins previously identified in our laboratory using yeast-two-hybrid screens.[unreadable] [unreadable] Little is known about the signals and processes that govern the trafficking of integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi and eventually to the plasma membrane. In a recent series of experiments we have identified a conserved 4 amino-acid hydrophobic trafficking motif near the C-terminus of NKCC1. We have studied the effects of various mutations of these amino acids by monitoring the levels of expression, complex glycosylation and aggregation of NKCC1 in transiently transfected HEK-293 cells. Our results indicate that mutation of any two these residues to alanine leads to a 60% reduction in complex glycosylation and increased NKCC1 aggregation, and that multiple mutations to alanine have cumulative effects. Mutation of this motif to AAAA reduces complex glycosylation by 90% and the level of NKCC1 expression by 70%, and leads to a marked increase in aggregation. This aggregated mutant protein was found to co-localize with an ER marker. This C-terminal hydrophobic motif is highly conserved across species and homologues in the NKCC1 gene family. Our results indicate that this motif is required for export of NKCC1 from the ER to the Golgi, and that retention of mutant proteins in the endoplasmic reticulum leads to their aggregation and destruction.